Functional Dynamics and Molecular Mechanism of Photolayse

光解酶是一种光酶，在许多生物体中利用蓝光的能量来逆转紫外线诱导的DMA损伤。人类缺乏这种酶，但最近对人类细胞系和小鼠的实验表明，通过基因治疗，光解酶可用于增强对阳光诱导的肿瘤发生的抵抗力。环丁烷嘧啶二聚体（CPD）和嘧啶-嘧啶酮（6-4）光产物是紫外线照射细胞产生的两大类具有细胞毒性、致突变性和致癌性的DNA光产物，是皮肤癌的主要诱因。一种假设的光解酶修复DNA的分子模型已经被提出并研究了很长时间，但直到去年我们使用超快光谱捕捉到催化中间体时，才观察到催化途径的直接证据。对原子尺度上非常有效的修复机制的理解一直缺乏。因此，本项目的第一个目标是在最基础的水平上揭示CPD光解酶修复DNA中二聚体的分子机制，确定其高修复效率的关键因素。目前对（6-4）光解酶修复(6-4)-光产物的机制研究较少，其修复动力学也没有CPD光解酶那么有效。因此，第二个目标是表征（6-4）光解酶的修复动力学，阐明其分子机制，并了解导致其效率低于CPD光解酶的因素。为了实现这些目标，我们整合了最先进的飞秒激光光谱，分子生物学方法和计算研究，并将修复过程的动态复杂性分解为基本步骤。利用飞秒时间分辨率和单残基空间分辨率，我们将揭示功能动力学的完整演变，并揭示光解酶修复DNA的基本机制。本研究将有助于合理的药物设计和基因治疗，以预防紫外线引起的皮肤癌。

DESCRIPTION (provided by applicant): Functional Dynamics and Molecular Mechanism of Photolyase Photolyase is a photoenzyme that uses the energy of blue light to reverse UV-induced DMA damage in many organisms. It is lacking in humans, but recent experiments in human cell lines and in mice indicate that through gene therapy, photolyase can be used to heighten resistence to sunlight-induced tumorigenesis. Cyclobutane pyrimidine dimer (CPD) and pyrimidine-pyrimidone (6-4) photoproduct are the two major classes of cytotoxic, mutagenic and carcinogenic DNA photoproducts produced by UV-light irradiation of cells and are the main cause of skin cancer. A hypothetical molecular model of DNA repair by photolyase has been proposed and investigated for a long time, but direct evidence for the catalytic pathway had never been observed until last year when we captured a catalytic intemediate using ultrafast spectroscopy. Understanding of the remarkably efficient repair mechanism at the atomic scale has been lacking. Thus, the first objective of the project is to reveal the molecular mechanism of dimer repair in DNA by CPD photolyase at the most fundamental level and determine the key factors for its high repair efficiency. The mechanism of the (6-4)-photoproduct repair by (6-4) photolyase has been less examined and the repair dynamics, less efficient than CPD photolyase, have never been explored. Therefore, the second objective is to characterize the repair dynamics of (6-4) photolyase, elucidate its molecular mechanism, and understand the factors contributing to its lessened efficiency compared to CPD photolyase. To achieve these goals, we integrate state-of-the-art femtosecond laser spectroscopy, molecular biology methods and computational studies, and break down the dynamic complexity of repair processes into elementary steps. With femtosecond temporal resolution and single-residue spatial resolution, we will unravel the complete evolution of the functional dynamics and lay bare the fundamental mechanisms of DNA repair by photolyase. The understanding gained from this work will help in rational drug design and gene therapy to prevent skin cancer induced by UV light.